Transcriptional repression of the oncofetal LIN28B gene by the transcription factor SOX6

The identification of regulatory networks contributing to fetal/adult gene expression switches is a major challenge in developmental biology and key to understand the aberrant proliferation of cancer cells, which often reactivate fetal oncogenes. One key example is represented by the developmental gene LIN28B, whose aberrant reactivation in adult tissues promotes tumor initiation and progression. Despite the prominent role of LIN28B in development and cancer, the mechanisms of its transcriptional regulation are largely unknown. Here, by using quantitative RT-PCR and single cell RNA sequencing data, we show that in erythropoiesis the expression of the transcription factor SOX6 matched a sharp decline of LIN28B mRNA during human embryo/fetal to adult globin switching. SOX6 overexpression repressed LIN28B not only in a panel of fetal-like erythroid cells (K562, HEL and HUDEP1; ≈92% p < 0.0001, 54% p = 0.0009 and ≈60% p < 0.0001 reduction, respectively), but also in hepatoblastoma HepG2 and neuroblastoma SH-SY5H cells (≈99% p < 0.0001 and ≈59% p < 0.0001 reduction, respectively). SOX6-mediated repression caused downregulation of the LIN28B/Let-7 targets, including MYC and IGF2BP1, and rapidly blocks cell proliferation. Mechanistically, Lin28B repression is accompanied by SOX6 physical binding within its locus, suggesting a direct mechanism of LIN28B downregulation that might contribute to the fetal/adult erythropoietic transition and restrict cancer proliferation.


CUT&RUN (LoV-U)
CUT&RUN was performed according to the LoV-U protocol described in (Zambanini et al., 2022).Per each sample 500,000 HUDEP1 (SOX6-Flag expressing) were harvested.) and the samples were incubated for 1 hour at 4°C.Beads were collected on the magnetic rack, while the supernatant was mixed in the corresponding collection tube, where it was cleaned up twice using Mag-Bind TotalPure NGS beads (Cat.#M1327, Omega Bio-Tek) at 2X, and then resuspended in 20 μl Tris-HCl pH 7.5.

Library preparation and sequencing
Library preparation was performed using the KAPA Hyper Prep Kit for Illumina platforms (Cat.#KK8504, KAPA Biosystems) according to the manufacturer's guidelines with the following modifications.End repair and A-tailing was performed with 0.4 size reactions with 20 μl of purified DNA.The thermocycler conditions were set to 12 °C for 15 min, 37 °C for 15 min and 58 °C for 25 min to prevent thermal degradation of the shortest fragments.Adapter ligation was done with 0.4 size reactions.KAPA Dual Indexed adapters were used at 0.15 μM.A post-ligation clean-up was performed with Mag-Bind TotalPure NGS beads at 1.2X.Resuspension was done in 10 mM Tris-HCl pH 8.0.Library amplification was performed with 0.5 size reactions.The cycling conditions were set as follows: 45 sec initial denaturation at 98 °C, 15 sec denaturation at 98 °C, 10 sec annealing/elongation at 60 °C, 1 min final extension at 72 °C, hold at 4 °C, with 13 cycles.After amplification, a post-amplification cleanup was performed with 1.2X beads.Libraries were then run on an E-Gel EX 2% agarose gel (Cat.# G402022, Invitrogen) for 10 min using the E-Gel Power Snap Electrophoresis System (Invitrogen).Bands of interest between 150 and 500 bp were cut out and purified using the QIAquick Gel Extraction Kit (Cat.#28706, QIAGEN) according to manufacturer's instructions.Libraries were quantified with the Qubit (Thermo Scientific) using their high sensitivity DNA kit (Cat #Q32854, Thermo Scientific), pooled and sequenced 36 bp pair-end on the NextSeq 550 (Illumina) using the Illumina NextSeq 500/550 High Output Kit v2.5 (75 cycles) (Cat.#20024906, Illumina).The CUT&RUN datasets (raw and processed files) have been deposited at ArrayExpress (https://www.ebi.ac.uk/arrayexpress/) under accession number E-MTAB-12800.

Suppl. Fig. 4 SOX6 5 LIN28B
directly represses LIN28B in Set-2 and Uke1 cells.Effect of SOX6 expression on LIN28B mRNA levels (a) and on representative Let-7 downstream targets (b).Suppl.Fig. gene expression in cancer.a, Expression of LIN28B from transcriptional profiling of human cancer cell lines of the indicated lineages (CCLE, Broad Institute) in DepMap.b, the dependency scores were calculated by Chronos (Dempster JM et al., 2021) for LIN28B CRISPR in CCLE lines shown in a and correlated to LIN28B gene expression levels in DepMap.c, Expression of LIN28B from transcriptional profiling of human cancer cell lines of the indicated tumor types (CCLE, Broad Institute) in DepMap.d, the dependency scores were calculated by Chronos.for LIN28B CRISPR in CCLE lines shown in (c) and correlated to LIN28B gene expression levels in DepMap.e, Expression of LIN28B and SOX6 from transcriptional profiling of human cancer cell lines of the peripheral nervous system, neuroblastoma (CCLE, Broad Institute) in DepMap.f, the dependency scores were calculated by Chronos for LIN28B CRISPR in the CCLE neuroblastoma cell lines shown in e and correlated to LIN28B gene expression levels in DepMap.a-f: TPM (Transcripts Per Million).b, d, f: LIN28 Gene Effect (Chronos).CRISPR (DepMap Public 23Q4+Score, Chronos).

Fig. 7 :
RNA expression of SOX6, LIN28B, MYC, and IGF2BP1 in cancer.a,b, data from Kildisiute et al., 2021.a, Single-cell RNA expression of SOX6, LIN28B, MYC, and IGF2BP1 in 16498 cells derived from human neuroblastoma (NBM) biopsies.Clusters were associated with 9 cell types according to the expression of marker genes.Samples were derived from pretreated tumors biopsies, only including viable areas.b, ranked scatterplot sorting cells based on their expression of SOX6 along the x-axis.The y-axis indicates the normalized expression of SOX6 (blue) and LIN28B (red).c,d, data from Lu et al., 2022 c, single-cell RNA expression of SOX6, LIN28B, MYC, and IGF2BP1 in cells derived from human hepatocellular carcinoma biopsies.Clusters were associated with 23 cell types according to the expression of marker genes.Samples were derived from primary and relapsed tumors and adjecent liver from 10 individuals.d, ranked scatterplot sorting cells based on their expression of SOX6 along the x-axis.The y-axis indicates the normalized expression of SOX6 (blue) and LIN28B (red).Suppl.Evolutionary conservation of the SOX6 consensus binding sites within the LIN28B locus, according to the UCSC genome browser (https://genome.ucsc.edu/).Human, mouse and chicken sequences are shown.Coordinates refer to the human assembly GRCh38/hg38.The sites order (from A to G) refers to the Lin28B locus map in Figure5.Suppl.Fig. 8 a, Venn diagram showing the number of peaks in the two replicates.The peaks present in both replicates are considered as reproducible, high-confidence events.b, Signal Enrichment plots for SOX6 showing common peaks from the two replicates.Signal entries in the heatmap are ordered by overall enrichment of the first profile on the left.c, Genomic coverage tracks obtained with CUT&RUN LoV-U targeting SOX6 representing the SOX6, PRMT1 and SLC6A9 loci and visualized with Integrative Genomic Viewer (IGV).From the top: two SOX6 replicates (SOX6 A and SOX6 B) with their respective negative control (anti-Flag).

Table 2 :
Kurita, R. et al.Establishment of immortalized human erythroid progenitor cell lines able to produce enucleated red blood cells.PLoSOne 8, e59890 (2013).list of antibodies and reagents used in this study 1